This invention is in the field of immunology and relates to compositions and methods for treating and diagnosing antiphospholipid (aPL) antibody-mediated pathologies. More specifically, the invention relates to conjugates of chemically-defined nonimmunogenic valency platform molecules and immunospecific analogs of aPL-binding epitopes as well as methods and compositions for producing these conjugates. Optimized analogs lack T cell epitopes. In addition, the invention relates to diagnostic assays for detecting the presence of and quantitating the amount of antiphospholipid antibodies in a biological sample. The invention also relates to a method of utilizing random peptide libraries to identify immunospecific analogs of aPL-binding epitopes.
Antiphospholipid antibodies occur in autoimmune diseases such as systemic lupus erythematosus (SLE) and antiphospholipid antibody syndrome (APS) as well as in association with infections and drug therapy. APS is characterized by one or more clinical features such as arterial or venous thrombosis, thrombocytopenia and fetal loss. APS may be primary or it may be associated with other conditions, primarily SLE. (Phospholipid-Binding Antibodies (Harris et al., eds., CRC Press, Boca Raton, Fla., 1991); McNeil et al. Advances in Immunology, Vol. 49, pp. 193-281 (Austen et al., eds., Academic Press, San Diego, Calif., 1991)). Approximately 30-40% of patients with SLE have aPL, however, 50% of patients with aPL antibodies do not have SLE. This 50% may have other autoimmune rheumatic diseases, miscellaneous conditions or they may have been subjected to drug therapy, particularly chlorpromazine. In one study of 70 patients, 26 males and 44 females, with primary APS (PAPS) but no evidence of SLE, the following features were observed: deep venous thrombosis (DVT) in 31; arterial occlusion in 31, particularly stroke or transient ischemia; myocardial infarctions in 15; recurrent fetal loss in 24; thrombocytopenia (TCP) in 32; 10 had a positive Coombs"" test; Evans"" syndrome in 7; anti-nuclear antibody (ANA) in 32, but less than 1:160 in 29; and antimitochondrial antibody (AMA) in approximately 24. (McNeil et al., supra.). Estimates vary but in about 5% of all stroke patients, aPL antibodies are thought to be an important contributing factor.
Transient aPL antibodies, such as those detected in a VDRL test, occur during many infections. Approximately 30% of patients possessing persistent aPL antibodies have suffered a thrombic event. The presence of aPL antibodies defines a group of patients within SLE who display a syndrome of clinical features consisting of one or more of thrombosis, TCP, and fetal loss. The risk of this syndrome in SLE overall is around 25%; this risk increases to 40% in the presence of aPL antibodies and decreases to 15% in their absence. Because aPL antibodies were thought to be directed at phospholipids in plasma membranes, it has been postulated that they may exert direct pathogenic effects in vivo by interfering with hemostatic processes that take place on the phospholipid membranes of cells such as platelets or endothelium. In patients with PAPS, the fact that aPL antibodies appear to be the only risk factor present is further evidence that these antibodies have a direct pathogenic role. Induction of PAPS by immunizing mice with human anticardiolipin antibodies is the best evidence yet that aPL antibodies are directly pathogenic (Bakimer et al. 1992 J. Clin. Invest. 89:1558-1563; Blank et al. 1991 Proc. Natl. Acad. Sci. 88:3069-3073).
Measurement of aPL antibodies in the clinical environment is still an imperfect art. A commercially available set of standard antisera (APL Diagnostics, Inc., Louisville, Ky.) allow generation of a standard curve for comparison of assays performed in various laboratories. A great deal of inconsistency exists, however, between the results obtained at these laboratories regarding the exact GPL and MPL, the unit of measurement for IgG and IgM antiphospholipid antibodies, respectively, ratings for given sera and the levels of GPL and MPL that are categorized as high, medium or low titer. The available commercial kits vary greatly in the values assigned to the commercially available standards (Reber et al. (1995) Thrombosis and Haemostat. 73:444-452). In spite of these limitations, there is general agreement that the epitopes recognized by antibodies in APS, PAPS and other aPL antibody-mediated diseases including recurrent stroke and recurrent fetal loss are located in the 5th domain of xcex22-GPI and are exposed to the antibody following binding of xcex22-GPI to cardiolipin.
It is now generally accepted that aPL antibodies recognize an antigenic complex comprised of xcex22-glycoprotein I (xcex22-GPI) and negatively-charged phospholipid, e.g., cardiolipin (McNeil et al. (1990) Proc. Natl. Acad. Sci. 87:4120-4124; Galli et al. (1990) Lancet 1:1544-1547) (hereinafter xe2x80x9caPL immunogenxe2x80x9d). xcex22-GPI is a minor plasma glycoprotein found free and in association with lipoprotein lipids where it is also known as apolipoprotein H (apo H). It consists of five independently folding domains referred to as Sushi or short consensus repeat domains that resemble similar domains in other proteins. xcex22-GPI has been reported to undergo antigenic and conformational changes upon binding phospholipid (Wagenkneckt et al. (1993) Thromb. Haemostas. 69:361-365; Jones et al. (1992) Proc. 5th Intl. Symp. Antiphospholipid Antibodies (Abstract S5)). The fifth domain contains the putative sites of lipid binding and aPL antibody binding (Hunt J. and S. Krilis, (1994) J. Immunol 152:653-659; Lauer et al. (1993) Immunol. 80:22-28). The pathological mechanism for aPL is unknown (McNeil et al., supra). Most explanations invoke endothelial cell function or platelet involvement (Haselaar et al. (1990) Thromb. Haemostas. 63:169-173). These explanations suggest that following blood vessel endothelial cell injury or platelet activation, the exposure or transbilayer migration of anionic phospholipid to the plasma-exposed surface may lead to xcex22-GPI-binding and trigger aPL antibody formation.
aPL antibodies may be directly prothrombotic by reducing prostacyclin formation (Vermylen, J. and J. Arnout (1992) J. Clin. Lab. Med. 120:10-12); by direct interference with the action of coagulation proteins; or by blocking the ability of xcex22-GPI to inhibit the intrinsic blood coagulation pathway, platelet prothrombinase activity, and ADP-mediated platelet aggregation (Arvieux et al. (1993) Thromb. Haemostas. 60:336-341).
A major new tool in medicinal chemistry in the search for lead compounds has been the advent of combinatorial libraries providing vast molecular diversity. Molecular diversity may arise from chemical synthesis or from biological systems (Scott., J. K. Rational Drug Design (CRC Press, Weiner, D. B. and W. V. Williams, eds., Boca, Raton, Fla., 1994); Moos et al. (1993) Ann. Reports Med. Chem. 28:315-324). By displaying random peptides on the surface of filamentous phage, epitope libraries containing hundreds of millions of clones for probing by clinically significant antibodies have been created (Scott, J. K. and G. P. Smith (1990) Science 249:286-390; Cesareni, G. (1992) FEBS Lett. 307:66-70). Such phage libraries are prepared by incorporating randomized oligonucleotide sequences into the phage genome, usually the pIII gene, which encode unique peptide sequences on the surface of each phage. Following sequential rounds of affinity purification and amplification, those phage that bind antibody are propagated in E. coli and the binding peptides identified by sequencing the corresponding coding region of viral DNA. In most cases, subsequent study will involve corresponding synthetic peptides after establishing their ability to bind antibody. Phage-based libraries have been used to mimic discontinuous epitopes (Luzzago et al. (1993) Gene 128:51-57; BaLass et al. (1993) Proc. Natl. Acad. Sci. 90:10638-10642). The potential plasma instability of peptide-based drugs has been successfully overcome by N-terminal blocking or by the judicious use of amino acid analogs (Powell, M. F. (1993) Ann. Reports Med. Chem. 28:285-293).
At present there is no selective, immunospecific therapy for patients showing high titers of aPL antibodies. In many cases use of drugs such as aspirin, steroids, and warfarin has proven to be largely inadequate (Phospholipid-Binging Antibodies (Harris et al., eds., CRC Press, Boca Raton, Fla., 1991); McNeil et al., supra). Synthetic mimetic peptides, characterized by (i) the inability to activate T cells while (ii) retaining the ability to bind immune B cells, are used to tolerize B cells in an antigen-specific manner. This technology is disclosed in co-owned, co-pending U.S. patent application, Ser. No. 08/118,055, filed Sep. 8, 1993, and U.S. Pat. No. 5,268,454, which are incorporated by reference herein in their entirety. As disclosed in the application and patent cited above, B cell tolerance entails administering such peptides conjugated to multivalent, stable, non-immunogenic valency platforms in order to abrogate antibody production via B cell anergy or clonal deletion after cross-linking surface immunoglobulin.
Although the exact molecular nature of the target epitopes recognized by aPL antibodies is unknown, the use of peptides derived from epitope libraries will allow for the construction of successful tolerogens. B cell tolerogens for the treatment of human systemic lupus erythematosus-related nephritis have also been disclosed in co-owned U.S. Pat. Nos. 5,276,013 and 5,162,515 which are incorporated by reference herein in their entirety.
This invention resides in the discovery of a method for identifying analogs of key epitopes recognized by aPL antibodies in patients suffering from PAPS, APS and other aPL antibody-mediated diseases, such as recurrent stroke and recurrent fetal loss, using random peptide phage libraries.
Accordingly, one aspect of the invention is an improved method for screening random peptide phage libraries in order to identify the peptide sequences which best mimic the epitopes recognized by aPL antibodies. This method comprises the steps of: (a) biopanning the library using methods modified from those known in the art; (b) eliminating very weakly-binding phage by micropanning the phage screened from step (a) by (i) incubating the phage in microplate wells coated with aPL antibody bound to Protein G, (ii) washing the microplate wells to remove unbound phage, (iii) eluting the bound phage, and (iv) infecting a microorganism such as E. coli with the eluted phage and counting the number of infected microorganisms by plating on agar; (c) determining the strongest-binding clones recovered in (b) by evaluation via phage-capture ELISA by (i) coating the wells of a microplate with aPL antibody, (ii) incubating the strongest-binding clones identified by micropanning in (b) in the coated wells and washing away unbound phage, (iii) quantitating the number of phage bound to the antibody using an enzyme-conjugated goat anti-phage antibody in a colorimetric ELISA assay and, if several equivalent strongly-binding clones are identified, an additional round of (d) phage-ELISA on the strongest-binding phage-capture-ELISA clone.
In this regard, the invention encompasses a method for identifying analogs of epitopes which specifically bind aPL antibodies isolated from humans suffering from an aPL antibody-mediated disease comprising: (a) preparing phage random peptide libraries; (b) screening said libraries with aPL antibodies to identify aPL mimetic epitopes, wherein said screening comprises (i) screening said libraries by biopanning; (ii) further screening phage isolated by biopanning in (i) by micropanning; and (iii) identifying phage containing aPL antibody high-affinity binding peptides recovered in (ii) by immunoassay.
The invention also encompasses a method of biopanning phage random peptide libraries to identify and isolate peptides which bind to aPL antibody comprising: (a) reacting affinity-purified aPL antibody with phage bearing random peptide inserts; (b) recovering phage bearing random peptide inserts which bind to the aPL antibody; (c) infecting a microorganism with phage recovered in (b); and (d) culturing the infected microorganism in an antibiotic-containing medium in order to isolate the phage.
The invention further encompasses a method of micropanning phage random peptide libraries to identify and isolate peptides having a high binding affinity to aPL antibodies comprising: (a) isolating phage bearing random peptide inserts by biopanning; (b) incubating the phage recovered in step (a) in microplate wells coated with aPL antibody bound to Protein G; (c) washing the microplate wells to remove unbound phage; (d) eluting bound phage; and (e) infecting a microorganism with phage recovered in (d); and (f) culturing the infected microorganism in an antibiotic-containing medium in order to isolate the phage.
The invention also encompasses the above method described wherein the immunoassay is a phage-capture ELISA comprising: (a) incubating phage bearing random peptide inserts isolated by micropanning in the microplate wells coated with aPL antibody; (b) washing away unbound phage;(c) incubating an enzyme-labeled anti-phage antibody to the wells; (d) washing away unbound enzyme-labeled anti-phage antibody; (e) adding a colorimetric substrate; and (f) measuring the absorbance of the substrate to identify high affinity-binding phage.
Also encompassed by the invention is the method described above and further comprising performing an additional phage-capture ELISA assay of the high affinity-binding phage comprising: (a) coating a uniform amount of the phage on microplate wells; (b) incubating aPL antibody in the wells; (c) washing away unbound antibody; (e) incubating an enzyme-labeled anti-aPL antibody with the bound aPL antibody; (f) washing away unbound enzyme-labeled anti-aPL antibody; (g) adding a colorimetric substrate to the wells; and (h) measuring the absorbance of the substrate to measure the relative binding affinity of the phage.
The invention also encompasses the method described above wherein the immunoassay is a colony-blot immunoassay comprising: (a) culturing a microorganism infected with phage bearing random peptide inserts on a nitrocellulose membrane atop an agar-containing culture medium; (b) replicate transferring the microorganism cultured in (a) by blotting the microorganism on a second nitrocellulose membrane atop an agar-containing culture medium; (c) incubating the transferred microorganism;(d) lysing the microorganism; (e) digesting the microorganism with lysozyme; (f) blocking the membrane with a gelatin solution; (g) incubating the membrane with aPL antibody; (h) washing away unbound aPL antibody; (i) incubating a enzyme-labeled anti-aPL antibody with the nitrocellulose membrane; (j) washing away unbound enzyme-labeled anti-aPL antibody; (k) adding a colorimetric substrate; and (l) measuring the absorbance of the substrate to identify high affinity-binding phage.
A method for assaying and ranking, for affinity-binding characteristics, epitopes which specifically bind aPL antibodies isolated from humans suffering from an aPL antibody-mediated disease is also encompassed, the method comprising: (a) coating wells of a microtitration plate with cardiolipin; (b) adding adult bovine or human serum as a source of xcex22-GPI to bind to the cardiolipin and to prevent non-specific binding to the wells of the plate; (c) incubating a solution of monomeric analog and a high-titered aPL antibody for a pre-determined time; (d) adding the aPL antibody/analog mixture to wells of the microtitration plate and incubating for a pre-determined time; (e) washing the wells to wash away unbound aPL antibody; (f) adding anti-human IgG conjugated with a label (e.g., an enzyme) to the wells of the plate and incubating for a pre-determined time; (g) washing the wells to wash away unbound anti-human IgG conjugate; (h) adding a substrate for the labeled conjugate and developing the substrate/label reaction for a pre-determined time;(i) measuring the end-product of the substrate/label reaction to quantitate the amount of aPL antibody bound to the well; (j) calculating the percentage inhibition, if any, of binding of the aPL antibody to determine the affinity of the analog to the aPL antibody.
Another aspect of the invention encompasses a fluorescence polarization peptide binding assay for determining the dissociation constants for peptides that bind to aPL antibodies. This assay detects direct binding of peptides to aPL antibodies.
The invention also encompasses a diagnostic immunoassay for determining the presence of aPL antibody in body fluids taken from subjects suspected of suffering from an aPL antibody-mediated disease comprising contacting a sample of a body fluid with an analog of an epitope which specifically binds aPL antibodies and determining by methods well known in the art whether aPL antibodies are present in the body fluid and, if present, quantitating the amount of aPL antibodies present in the fluid. One such immunoassay comprises: (a) coating wells of a microtitration plate with an analog of an epitope which specifically binds aPL antibodies; (b) washing the wells to wash away unbound analog; (c) adding a test sample of a body fluid to the wells and incubating for a pre-determined time; (d) washing the wells to remove unbound test sample; (e) adding anti-human IgG conjugated with a label to the wells of the plate and incubating for a pre-determined time;(f) washing the wells to wash away unbound anti-human IgG conjugate; (g) adding a substrate for the labeled conjugate and developing the substrate/label reaction for a pre-determined time; (h) measuring the end-product of the substrate/label reaction to determine the presence of anti-aPL antibody in the test sample. A diagnostic immunoassay as described above wherein the immunoassay is quantitative is also encompassed.
The phage-ELISA assay consists of (i) coating a uniform amount of different clones on wells of a microtitration plate followed by (ii) identifying the peptide inserts which most strongly bind aPL antibody by adding antibody to the wells and developing the reaction with an enzyme-labeled anti-human IgG conjugate. The random peptides displayed by the phage which have a high binding affinity to aPL antibody as measured by phage-ELISA, colony blot or phage-capture-ELISA represent the analogs of the aPL-specific epitope. These peptides are then synthesized and ranked for strength of binding using competition assays.
Another aspect of the invention is aPL antibody-binding analogs that bind specifically to B cells to which an aPL epitope binds. Optimized analogs lack T cell epitope(s).
Yet another aspect of the invention is a composition for inducing specific B cell tolerance to an aPL immunogen comprising a conjugate of a nonimmunogenic valency platform molecule and an aPL antibody-binding analog that (a) binds specifically to B cells to which an aPL immunogen binds and (b) lacks T cell epitope(s).